AN alpha -GALACTOSIDASE ENZYME

Abstract: A DNA construct comprising a DNA sequence encoding a polypeptide having alpha -galactosidase activity, wherein the DNA sequence a) encodes a polypeptide comprising the amino acid sequence shown in the appended SEQ ID No. 3, or b) is an analogue of the DNA sequence of a), which i) hybridizes with the DNA sequence shown in the appended SEQ ID No. 1 or 2 or an oligonucleotide probe prepared on the basis of said DNA sequence or on the basis of the amino acid sequence shown in SEQ ID No. 3 under the conditions defined herein; ii) encodes a polypeptide reactive with an antibody reacting with at least one epitope of a polypeptide comprising the amino acid sequence shown in the appended SEQ ID No. 3, and/or; iii) encodes a polypeptide being at least 50 % identical with the polypeptide having the amino acid sequence shown in the appended SEQ ID No. 3, as well as an alpha -galactosidase enzyme encoded by the DNA construct.

Description: AN α-GALACTOSIDASE ENZYME FIELD OF THE INVENTION The present invention relates to a DNA construct encoding an α-galactosidase enzyme and variants thereof having α-galacto¬ sidase activity, a recombinant expression vector and a cell harbouring said DNA construct, and a method of preparing an α-galactosidase enzyme preparation by use of recombinant DNA techniques. The α-galactosidase enzyme encoded by the DNA construct of the invention may, inter alia, be used for the degradation of α-galactosides present in various plant prod¬ ucts, or as a digestive aid. BACKGROUND OF THE INVENTION α-galactosidase is a well-known enzyme involved in the hydro- lysis of α-galactosides present in, for instance, various im¬ portant plants or plant parts used for nutritional purposes such as legumes, vegetables, grains, cereals and the like, α- galactosidase enzymes are produced by various microorganisms, plants and animals. Mammals, however, are deficient in intes- tinal α-galactosidase production and, consequently, are inca¬ pable of decomposing ingested α-galactosides by themselves. Instead, ingested α-galactosides are decomposed by microorga¬ nisms present in the intestine. This microbial decomposition normally results in flatulence and further confers a diges- tive discomfort to the mammal upon ingestion of α-galactosi- de-containing food or feed. The physiological effects of α- galactosides are discussed in detail by Rackis, J. J. , 1975. In order to overcome the problem associated with mammalian α- galactosidase deficiency, α-galactosides contained in food or feed have been modified prior to ingestion, for instance en- zy atically by the action of α-galactosidase. Alternatively, α-galactosidase has been suggested as a digestive aid, cf. WO 90/14101. The production of α-galactosidase has been reported from bac- teria, e.g. Bacillus εtearothermophilus (US 3,846,239), yeasts, e.g. Saccharomyces cereviciae (US 4,431,737), fungi, e.g. strains of the genii Neurospora and Rhizopus (Worthing- ton and Beuchat, 1974) , Aspergillus oryzae (Cruz and Park, 1982) , A. ficuum (morphologically similar A. niger) (Zapater et al., 1990) and A. niger (Bahl and Agrawal (1969 and 1972), Christakopoulos et al. (1990) , Chun and Lee (1988) , Jung and Lee (1986) , Lee and Wacek (1970) , Adya and Elbein (1976) , Ka- neko et al. (1991)). All of these references, however, de¬ scribe the α-galactosidase production by conventional fermen- tation of naturally occurring or mutated microbial strains. Overbeeke et al., 1990, describes the production of α-galac¬ tosidase from guar in Bacillus subtilis and Aslandis et al, 1989, describes an α-galactosidase from E. coli . An A. niger α-galactosidase enzyme preparation (Alpha-Gal™) produced by conventional fermentation is available from Novo Nordiεk A/S, Denmark. One drawback associated with the pro¬ duction of α-galactosidase by fermentation of A. niger is that substantial amounts of oxalic acid, an undesired by-pro¬ duct, are produced by A. niger simultaneously with the pro¬ duction of α-galactosidase. It would be desirable to be able to produce an A. niger α-ga- lactosidase enzyme preparation with reduced or without simu¬ ltaneous production of oxalic acid, and further to increase the yield and the purity of the α-galactosidase preparation so produced. The object of the present invention is to device means and methods for the production of α-galactosidase enzymes by re¬ combinant DNA techniques. By use of such techniques it is contemplated to be possible to produce α-galactosidase in substantially larger amounts and more economical than what is possible by use of conventional fermentation technology and at the same time avoid or reduce the amount of oxalic acid formed. BRIEF DESCRIPTION OF THE INVENTION Accordingly, in a first aspect the present invention relates to a DNA construct comprising a DNA sequence encoding a poly¬ peptide having α-galactosidase activity, wherein the DNA se¬ quence a) encodes a polypeptide comprising the amino acid sequence shown in the appended SEQ ID No. 3, or b) is an ana¬ logue of the DNA sequence of a) , which i) hybridizes with the DNA sequence shown in the appended SEQ ID No. 1 or 2 or an oligonucleotide probe prepared on the basis of said DNA sequence or on the basis of the amino acid sequence shown in SEQ ID No. 3 under the conditions defined below, and/or ii) encodes a polypeptide reactive with an antibody reacting with at least one epitope of a polypeptide comprising the a- mino acid sequence shown in the appended SEQ ID No. 3, and/or iii) encodes a polypeptide being at least 50% identical with the polypeptide having the amino acid sequence shown in the appended SEQ ID No. 3. The nucleotide sequence shown in SEQ ID No. 1 illustrates an entire α-galactosidase gene (including introns) isolated and characterized from a strain of Aspergilluε niger, and the nu¬ cleotide sequence shown in SEQ ID No. 2 is the corresponding cDNA sequence. The nucleotide sequences are further described in the examples hereinafter. The amino acid sequence shown in SEQ ID No. 3 is deduced from the DNA sequence shown in SEQ ID No. 2 and illustrates the amino acid sequence of the A. niger α-galactosidase enzyme including its signal peptide. In a further aspect the present invention relates to a recom¬ binant expression vector harbouring the DNA construct of the invention and a cell which either harbours the DNA construct or the expression vector of the invention. A still further aspect of the present invention is a process for the production of a polypeptide exhibiting α-galactosid¬ ase activity, which process comprises culturing a cell as de¬ scribed above harbouring a DNA construct of the invention in a suitable culture medium under conditions permitting expres¬ sion of the polypeptide, and recovering the resulting poly¬ peptide from the culture. The polypeptide exhibiting α-galactosidase activity may com- prise the amino acid sequence shown in SEQ ID No. 3. or be a variant thereof. The variant may be a naturally-occurring variant derived from any source or organism, and in particu¬ lar from a naturally-occurring microorganism or a mutant or derivative thereof. Furthermore, the "variant" may be a gen- etically engineered variant, e.g. prepared by suitably mod¬ ifying a DNA sequence of the invention resulting in the addi¬ tion of one or more amino acid residues to either or both the N- and C-terminal end of the polypeptide encoded by the unmo¬ dified DNA sequence, substitution of one or more amino acid residues at one or more different sites in the amino acid se¬ quence, deletion of one or more amino acid residues at either or both ends of the polypeptide or at one or more sites in the amino acid sequence, or insertion of one or more amino a- cid residues at one or more sites in the amino acid sequence. By use of the process of the invention it is possible to pro¬ duce enzyme preparations having a higher content of α-galac¬ tosidase than what is possible by conventional fermentation of a parent microorganism, such as A . niger, inherently pro- ducing the α-galactosidase. Furthermore, the resulting α-ga¬ lactosidase preparations are essentially free from any other components derived from the parent microorganism, in particu¬ lar components giving rise to undesirable enzymatic side-ac- tivities. Accordingly, by use of the process of the invention it is possible to optimize the production of α-galactosidase enzyme components thereby producing an enzyme preparation with a higher specific α-galactosidase activity at lower cost than what is possible by methods known in the art. At the same time the undesirable production of oxalic acid may be substantially reduced or avoided. DETAILED DISCLOSURE OF THE INVENTION In the DNA construct of the invention, the analogue of the DNA sequence encoding a polypeptide having α-galactosidase activity may, for instance, be a subsequence of said DNA se- quence, a genetically engineered modification of said sequen¬ ce which may be prepared by well-known procedures, e.g. by site-directed mutagenesis, and/or a DNA sequence isolated from another organism and encoding an α-galactosidase enzyme with substantial similarity to the α-galactosidase having the amino acid sequence shown in SEQ ID No. 3. The actual sequen¬ ce of the analogue is not critical as long as the analogue has at least one of the properties i)-iii) listed above. These properties are further discussed below. Property i) , i.e. the hybridization of a DNA sequence with the DNA sequence shown in the SEQ ID No. 1 or 2 or with a suitable oligonucleotide probe prepared on the basis of said DNA sequences or on the basis of the polypeptide shown in SEQ ID No. 3 may be carried out under any suitable conditions al- lowing the DNA sequences to hybridize. For instance, 1 μg of total DNA expected to harbour an analogous DNA sequence is subjected to complete digestion with, e.g. EcoRI, BamHI or Hindlll, and applied to a 1% agarose gel. The DNA fragments are separated by electrophoresis, and then transferred to an Immobilon™-N membrane (Millipore Corporation) following the Manufacturers instructions. The membrane is prehybridized following the manufacturers instructions and then the DNA sequence shown in SEQ ID No. 1 or 2 or a representative frag- ment thereof, labelled with 32 p by primer extension (Sambrook et al., 1989), is added as a probe, and the temperature re¬ duced to 45°C. After 18 hrs of hybridization the membrane is washed repeatedly in 6xSSC, 0.1% SDS at 45°C. The membrane is then subjected to autoradiography and evaluated. Property ii) , i.e. the immunological cross reactivity may be assayed using an antibody raised against or reactive with at least one epitope of the α-galactosidase enzyme comprising the amino acid sequence shown in SEQ ID No. 3. The antibody, which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. The immunological cross-reactivity may be determi¬ ned using assays known in the art, examples of which are We- stern Blotting or radial immunodiffusion assay, e.g. as de¬ scribed by Hudson et al., 1989. Property iii) may be determined by comparing the amino acid sequences of the polypeptide encoded by the analogue and the polypeptide sequence shown in SEQ ID No. 3 by use of well- known algorithms, such as the one described by Lip an and Pearson (1985) . In the present context, "identity" is used in its conventional meaning, i.e. intended to indicate the num¬ ber of identical amino acid residues occupying similar posi- tions in the two (or more) amino acid sequences to be com¬ pared. It is believed that an identity of above 50% such as above about 70%, 75%, 80%, 90% and in particular above about 95% with the amino acid sequence shown in SEQ ID No. 3 is indica¬ tive for homology with the α-galactosidase encoded by the DNA sequences shown in SEQ ID Nos. 1 and 2. From an alignment study of the amino acid sequence shown in SEQ ID No. 3 and the amino acid sequence encoding the E. coli α-galactosidase disclosed by Aslandis et al., 1989 an identity of about 30% was found. As far as the present inventors are aware this is the only α-galactosidase with a known amino acid sequence that show any comparable identity to the α-galactosidase en¬ coded by the DNA construct of the invention. It is well known that homology exists between polypeptides of different origins, and α-galactosidases homologous to α-ga- lactosidases from yeast have been found in plants as well as in mammals. Analogously herewith, it is contemplated that in the DNA construct of the invention, the DNA sequences may be derived from an animal including a mammal and an insect, a plant or a microorganism. In the present context, especially interesting origins are bacteria and fungi. The term "fungi" is intended to include yeasts and filamentous fungi. As stated above, the DNA sequences shown in SEQ ID Nos. 1 and 2 encoding an α-galactosidase are derived from a fungus, more particularly from A. niger. It is contemplated that other fun¬ gal α-galactosidases may show a substantial homology, either on the DNA or amino acid level, with the A. niger α-galacto¬ sidase disclosed herein, and accordingly, DNA sequences of the DNA construct of the invention may be derived from a fun¬ gus, in particular from a strain of Aspe_rgi22us such as from a strain of A. niger. An example of such strain is the strain of A. niger deposited with the American Type Culture Collec¬ tion under the number ATCC 16882. When isolated from A. niger the α-galactosidase enzyme is contemplated to exist as a number of isoenzy es, presumably due to heavy glycosylation. It is expected that the α-galac¬ tosidase encoded by the DNA construct of the invention may be in the form of different isoenzymes, depending on the circum¬ stances under which it is produced, and in particular on the host cell in question producing the enzyme. In Example 1 below characteristic properties are described of an A. niger α-galactosidase enzyme (as isolated from A. niger) . It has surprisingly been found that some properties of an α-galactosidase expressed from a DNA construct of the invention differ from the corresponding properties of the α- galactosidase isolated from A. niger. Thus, whereas the isolated α-galactosidase has a pH optimum in the range of 3.8-6.0, the α-galactosidase expressed from the DNA sequence shown in SEQ ID No. 2 in an Aspergillus ory- zae host cell has been found to have a pH optimum in the ran¬ ge of 5.0-7.0 (cf. Example 5 herinafter) . Based on the corresponding properties of the purified A. nig¬ er α-galactosidase, it is contemplated that an α-galactosid¬ ase enzyme encoded by the a DNA construct of the invention has a pi in the range of 4.0-5.0 (depending on the isoenzyme in question) such as about 4.3 as determined by IEF as de¬ scribed herein, a temperature optimum within the range of 50- 70°C, a molecular weight of about 170 kDa, and/or a specific activity of above about 250 GALU/mg protein. 1 GALU is the unit of α-galactosidase strength which is further defined in the materials and methods section below. It will be understood that the preferred DNA construct of the invention is one, in which the DNA sequence is as shown in the appended SEQ ID No. 1 or 2. The DNA sequence of the DNA construct of the invention may be isolated by well-known methods. Thus, the DNA sequence may, for instance, be isolated by establishing a cDNA or genomic library from an organism expected to harbour the sequence, e.g. a cell of any of the origins mentioned above, and scree- ning for positive clones by conventional procedures. Examples of such procedures are hybridization to oligonucleotide pro¬ bes synthesized on the basis of the full or partial amino acid sequence of the A . niger α-galactosidase comprising the amino acid sequence shown in SEQ ID No. 3 in accordance with standard techniques (cf. Sambrook et al., 1989), and/or se¬ lection for clones expressing an appropriate biological acti¬ vity as defined above, and/or selection for clones producing a protein which is reactive with an antibody raised against the A. niger α-galactosidase. A preferred method of isolating a DNA construct of the inven- tion from a cDNA or genomic library is by use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes prepared on the basis of the amino acid sequence shown in SEQ ID No. 3. For instance, the PCR may be carried out using the techniques described in US Patent No. 4,683,202 or by R.K. Saiki et al. (1988). Alternatively, the DNA sequence of the DNA construct of the invention may be prepared synthetically by established stan¬ dard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers (1981) , or the method described by Matthes et al. (1984). According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automa¬ tic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. Finally, the DNA construct may be of mixed genomic and syn¬ thetic, mixed synthetic and CDNA or mixed genomic and CDNA origin prepared by ligating fragments of synthetic, genomic or CDNA origin (as appropriate) , the fragments corresponding to various parts of the entire recombinant DNA molecule, in accordance with standard techniques. As stated above, the DNA construct of the invention may also comprise a genetically modified DNA sequence. Such sequence may be prepared on the basis of a genomic or cDNA sequence of the invention, suitably modified at a site corresponding to the site(s) of the polypeptide at which it is desired to in¬ troduce amino acid substitutions, e.g. by site-directed mut- agenesis using synthetic oligonucleotides encoding the de- sired amino acid sequence for homologous recombination in accordance with well-known procedures, or by use of random mutagenesis, e.g. through radiation or chemical treatment. Examples of suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide, but which may corre¬ spond to the codon usage of the host organism into which the recombinant DNA molecule is introduced (i.e. modifications which, when expressed, results in e.g. an α-galactosidase comprising the amino acid sequence shown in the appended SEQ ID No. 3), or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different polypeptide structure without, however, impairing properties of the polypeptide such as enzymatic properties thereof. Other examples of possible modifications are inser¬ tion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence and deletion of one or more nucleotides at either end of or with¬ in the sequence. The recombinant expression vector carrying the DNA construct of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously repli¬ cating vector, i.e. a vector which exists as an extrachromo- somal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid or a bacteriophage. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding prot- eins either homologous or heterologous to the host cell. For instance, examples of suitable promoters for directing the transcription of the DNA construct of the invention in a fungal host cell are the TAKA promoter and the triose phos- phate isomerase promoter of Aspergillus oryzae, the amylogly- cosidase promoter and the glyceraldehyde-3-phosphate dehydro- genase promoter of Aspergillus niger and the cellobiohydrola- se I promoter of Trichoderme reseei . The expression vector of the invention may also comprise a suitable terminator operably connected to the DNA construct of the invention. The terminator is suitably derived from the same source as the promoter of choice. The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B.subtilis or B.licheniformis. or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. While intracellular expression may be advantageous in some respects, e.g. when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In order to obtain extracellular expression, the expression vector should normally further comprise a DNA sequence encod¬ ing a preregion, i.e. a signal peptide, permitting secretion of the expressed α-galactosidase or a variant thereof into the culture medium. The procedures used to ligate the DNA construct of the inven¬ tion, the promoter, terminator and other elements, respect¬ ively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al. (1989)). The cell of the invention either comprising a DNA construct or an expression vector of the invention as defined above is advantageously used as a host cell in the recombinant produc¬ tion of a polypeptide of the invention. The cell may be tran- sformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromo- some may be performed according to conventional methods, e.g. by homologous recombination. Alternatively, the cell may be transformed with an expression vector as described below in connection with the different types of host cells. The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell which, on cultivation, produces large amounts of the poly¬ peptide. Examples of suitable bacteria are grampositive bacteria such as Bacillus subtilis. Bacillus licheniformis. Bacillus lent- us, Bacillus brevis. Bacillus stearothermophilus, Bacillus alkalophilus. Bacillus amyloliquefaciens. Bacillus coagulans. Bacillus circulans. Bacillus lautus, Bacillus thurinqiensis or Streptomyces lividans. Streptomyces urinus, or gra nega- tive bacteria such as E.coli. The transformation of the bac¬ teria may for instance be effected by protoplast transforma¬ tion or by using competent cells in a manner known per se. The yeast organism may favourably be selected from a species of Saccharomvces or Schizosaccharomvces. e.g. Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus. e.g. Aspergillus orvzae or Aspergillus niger. Fungal cells may be transformed by a pro¬ cess involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host organ¬ ism is described in, e.g., EP 238 023. In a yet further aspect, the present invention relates to a method of producing a polypeptide of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the polypep¬ tide and recovering the polypeptide from the cells and/or culture medium. The medium used to cultivate the cells may be any convention¬ al medium suitable for growing the host cell in question. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalo- gues of the American Type Culture Collection) . The polypeptide may be recovered from the medium by conven¬ tional procedures including separating the cells from the me¬ dium by centrifugation or filtration, if necessary after dis- ruption of the cells, precipitating the proteinaceous compo¬ nents of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like, the actual recovery method being dependant on the kind of polypeptide in question as well as the desired final purity thereof. Depending on the degree of purification of the polypeptide produced by the process of the invention, the resulting po- lypeptide preparation may contain minor amounts of other en¬ zymatic components inherently produced by the host cell used for the production. For instance, when a fungal cell, such as one of the genus Aspergillus, is used as a host cell for the production of a recombinant fungal α-galactosidase enzyme, certain of the enzymatic side-activities normally found in α- galactosidase preparations produced by conventional fermenta¬ tion of a parent fungal strain may also be produced and re¬ covered together with the recombinant polypeptide produced in accordance with the present invention. An example of an enzy¬ me normally found in α-galactosidase preparations prepared by conventional techniques is the enzyme invertase. This enzyme is inherently produced by a number of Aspergillus strains and consequently may also be found in α-galactosidase prepara¬ tions produced by Aspergillus strains in accordance with the present invention, although in a considerably lower amount as compared to the α-galactosidase than what is observed in con¬ ventional fermentation. Thus, in the context of the present invention, a substantial increase in the ratio of α-galacto¬ sidase to other enzymatic activities may be obtained in addi¬ tion to the increased total yield of α-galactosidase. If it is desired to produce substantially pure α-galactosi- dase or alternatively α-galactosidase preparation free from certain undesired enzymatic side-activities (an example of which - for some uses of the α-galactosidase - is invertase) one may either remove the side-activity(ies) by purification or one may choose a production organism incapable of produc- ing the side-activity(ies) in question. The α-galactosidase encoded by the DNA construct of the in¬ vention may be used for a number of purposes involving hydro¬ lysis of α-galactosides to galactoses and sucroses. For instance the α-galactosidase preparation encoded by the DNA construct and produced by the process of the invention may be used for the hydrolysis of α-galactosides present in, e.g., plants or plant parts which, for instance, are intended for nutrition of mammals or for fermentation of microorgan¬ isms. As indicated above, such plants and plant parts com¬ prise legumes such as peas and beans, nuts, seeds, grains, cereals and vegetables including potatoes, beets and chicory, as well as processed products thereof including flour, meal, bran, molasses, etc. Thus, the α-galactosidase enzyme pre¬ pared according to the invention may be used for the pre- treatment of food or feed containing α-galactosides and for modification of soy bean or sugar beet molasses used as a substrate in the fermentation of microorganisms. One important use of the α-galactosidase preparation prepared according to the invention is in the modification of soy beans or soy products such as soy bean molasses, soy bean sauce, soy bean milk, and soy bean whey. Accordingly, in a further aspect the present invention re- lates to a method of preparing an enzyme-modified soy bean product comprising subjecting a composition containing the soy bean product to be modified to enzymatic treatment in the presence of an α-galactosidase preparation produced according to the invention. The enzymatic treatment may be performed by use of methods known in the art. For instance, soy bean meal may be modified by suspending the soy bean product in water so as to obtain a dry matter content in the resulting suspen¬ sion of about 15-20%, adjusting pH to about 4.5-6 and trea¬ ting the resulting suspension with 0.5% of an α-galactosidase preparation of the invention comprising about 500 GALU/g for 2-8 hours at 50°C. The resulting modified product may subse¬ quently be spraydried. Furthermore, soy bean products may be produced as described by Olsen et al., 1981 and Eriksen, 1983, and the α-galactosidase preparation may be added, when appropriate, during the production. In the preparation of soy milk the α-galactosidase preparations may be added to the ex¬ tract resulting after separation of solid particles from the soy bean material or during evaporation or in the final con¬ centrated soy milk product. Alternatively, a soy bean product may be treated by a method comprising a) inserting a DNA construct of the invention encoding an α-galactosidase, optionally present in a suitable express¬ ion vector, into a suitable host organism, b) culturing the host organism in a suitable culture medium under conditions permitting expression of the polypeptide encoded by the DNA construct, and recovering the resulting polypeptide from the culture, and c) subjecting a composition containing the soy bean pro¬ duct to be modified to enzymatic treatment in the presence of the polypeptide recovered in step b) . Step a) and b) may be performed as disclosed herein. The α-galactosidase preparation produced according to the invention may further be used for the production of sugar from sugar beets in accordance with well-known procedures to improve the sugar yield by hydrolysing raffinose and stacchy- ose to galactose or sucrose. Another important use of the α-galactosidase prepared accord¬ ing to the invention is for the .in vivo conversion of α-ga- lactoside-linked sugars in mammals, e.g. as described in WO 90/14101. The α-galactosidase preparation may thus be used as digestive aid. For this purpose the α-galactosidase preparation may be combined with a suitable carrier or excipient so as to be in the form of a tablet, a capsule, a powder, a liquid, or in a soft-gel capsule form. The amount of α-galactosidase present in such formulations is in the range of 500-20000 GALU/G. In a further aspect the present invention relates to a food or feed comprising an α-galactosidase preparation prepared according to the invention. The α-galactosidase preparation is typically included in an amount corresponding to about 1- 20 GALU/g of food or feed. Examples of food or feed in which the α-galactosidase preparation may be included is given above. The present invention is described in the following by refer¬ ence to the appended drawings, in which Fig. 1 illustrates the construction of pCaHj 413 as described in Example 4, Fig. 2 illustrates the construction of pCaHj 414 as described in Example 4, Fig. 3 illustrates the construction of pCaHj 424 as described in Example 4, Fig. 4 illustrates the pH optimum of α-galactosidase, Fig. 5 is a HPLC chromatogram illustrating the degradation of raffinose by α-galactosidase, and Fig. 6 is a HPLC chromatogram illustrating the degradation of stacchyose by α-galactosidase. The present invention is further illustrated in the following examples, which are not, in any manner, intended to limit the invention as disclosed herein. MATERIALS AND METHODS Starting material The α-galactosidase preparation used in the following examples is a commercial A. niger α-galactosidase preparation (Alpha-Gal™, Batch KAN 0001) available from Novo Nordisk A/S, Denmark. Determination of α-Galactosidase Activity (GALU) 1 GALU is defined as the amount of α-galactosidase required for hydrolyzing 1 μmole p-nitrophenyl α-D-galactopyranoside (to p-nitro phenol + galactose) in one minute under the fol¬ lowing conditions: Substrate: 0.80 mM p-NPGal pH: 5.5 - acetate buffer 0.0333M Temperature: 37°C Reaction time: 15 min. Reagents : 1. BUFFER: Acetate buffer 0.05 M, pH 5.5 2 . SUBSTRATE: 1.2 mM p-Nitrophenyl-α-D-galactopyranoside 3. STOP REAGENT: Borax - NaOH buffer 0.0625 M, pH 9.7 4. COLOUR STANDARD: 4-Nitrophenol, 240 μM Procedure A standard curve is prepared by mixing 2 ml of substrate and 1 ml of various dilutions of colour standard (prepared with demineralized water) and adding 5 ml of stop reagent. When making the colour standard blank use demineralized water instead of colour standard. Measure OD 405 . Weigh and dilute the enzyme preparation to a concentration corresponding to an activity of about 0.0015 GALU/ml. Calculation of Activity: Make the colour standard curve (AOD against concentration) . The activity is calculated according to the following for¬ mula: (A s A„) 10 - 3 Act M where A s = The reading on the standard curve in μM 4-NP, corre¬ sponding to OD 405 for the sample. 5 A B = The reading on the standard curve in μM 4-NP, corre¬ sponding to OD 405 for the sample blank. F s = Dilution factor for the sample. T = Reaction time in minutes (= 15) . M = Amount of sample weighed out. 10 10 "3 = Conversion factor 1/ml. Fed batch fermentation Fed batch fermentation was performed in a medium comprising maltodextrin as a carbon source, urea as a nitrogen source 15 and yeast extract. The fed batch fermentation was performed by innoculating a shake flask culture of A. oryzae host cells in question into a medium comprising 3.5% of the carbon source and 0.5% of the nitrogen source. After 24 hours of cultivation at pH 7.0 and 34°C the continuous supply of addi- 20 tional carbon and nitrogen sources were initiated. The carbon source was kept as the limiting factor and it was secured that oxygen was present in excess. The fed batch cultivation was continued for 4 days, after which the enzyme could be recovered by centrifugation, ultrafiltration, clear filtra- 25 tion and germ filtration. Characterization of an enzyme of the invention pH optimum is measured by using 2mM PNP-α-galactosidase in 0.1 M citrate/phosphate buffers pH 2.5-10 as a substrate. To 30 0.5 ml substrate is added 10 μl enzyme solution (lOOx diluted in 3 mg/ml BSA) , the mixture is incubated at 30°C for 15 min¬ utes and the enzyme is heat-inactivated at 95°C. Three samples and one blank are prepared. 100 μl are pipetted into a microtiter plate well, 100 μl 1M tris buffer pH 7.0 are 35 added and the absorbance is measured in the microtiter reader at 405 nm. Paranitrophenol is used as a standard. The speci¬ fic activity at the optimal pH is calculated. Temperature stability is measured by leaving the enzyme sol¬ ution (in BSA or in 0.25% raffinose) at different tempera¬ tures for 1 and 2 hours before incubations are carried out at optimal pH in PNP-α-galactoside. Measurements are carried out as above. Specific activity towards raffinose is measured by carrying out incubations at optimal pH at different raffinose concen¬ trations (2-32 mM) . Released galactose is determined by the amount of reducing sugars. Reducing sugars are determined by reaction, in microtiter plates, with a PHBAH reagent comprising 0.15 g of para hydroxy benzoic acid hydrazide (Sigma H-9882) , 0.50 g of po- tassium-sodium tartrate (Merck 8087) and 2% NaOH solution up to 10.0 ml. Results of blanks are subtracted. In order to test for activity towards raffinose and stacchy- ose with and without the presence of galactose and sucrose, solutions are mixed according to the table below. The buffer is 0.1 M acetate buffer at the optimal pH for each enzyme. 10 μl of enzyme solution (diluted 10 times) is added and incuba¬ tions are carried out at 30°C for 0, 1, 2, 4 and 24 hours. 25 μl of the supernatant is analysed on the Dionex HPLC system (PAl column, 0.12 M NaOH eluent, 1 ml/min flow rate, Pulsed Amperometric Detection) which separates all the saccharides. This experiment should also reveal if any transferase activ¬ ity can be ascribed to the α-galactosidases. EXAMPLE 1 Purification and characterization of α-galactosidase from Aspergillus niger Salt precipitation A sample of the α-galactosidase preparation was washed with 5 volumes of ionwater in an Amicon-UF-cell (membrane GR 60PP, Cut Off 25.000). Salt precipitation was achieved by use of (NH 4 ) 2 S0 4 at 60% saturation (385 g/1) , at which degree of sat¬ uration α-galactosidase had been shown to precipitate. The (NH 4 ) 2 S0 4 was added slowly (more than one hour) under stirring at room temperature. The pH was kept constant at pH 5.5 by addition of a base. The precipitate was redissolved in water and washed in an Amicon-UF-cell (membrane GR 60 PP) until a conductance of about 0.9 mS was reached. Ionexchange The redissolved and washed precipitate was subjected to an- ionexchange on a DEAE-Sepharose-CL-6B column equilibrated with a citrate/phosphate buffer, pH 5.5 (0.002 M citric acid/0.006 M Na 2 HP0 4 ) , and a conductivity of about 0.9 mS. The α-galactosidase was eluted with 0-0.5 M NaCl and fractions containing α-galactosidase activity were pooled. Gelfiltration The pooled α-galactosidase fractions were concentrated 10 x to obtain a protein content of about 16 mg/ml. The gelfiltra¬ tion was performed on a Sephadex G100 (Mw 4.000-150-000) gel- filtration column equilibrated with the buffer specified above. The α-galactosidase which was present in the front fraction and contained 5.6 mg of protein, was subsequently analysed for purity by use of the IEF Phast system and the SDS-PAGE Phast system as described below. The specific activity of the front fraction was deter ed to 264 GALU/ g Protein as described above. The protein content was determined spectrophoto etrically at 280 n . IEF The α-galactosidase fraction was run on an IEF-PAA pH 4-6.5 (Pharmacia Phast System File Nos. 100 and 200) . A strong bond could be observed at pH 4.3 and a weak shadow was observed at pH 4.2. It was concluded that the pi of the purified enzyme was 4.3. SDS-PAGE The α-galactosidase fraction was run on a SDS-gradient gel PAA 10-15 (Pharmacia Phast System as above) . Before the sample was loaded the protein was subjected to denaturation and reduction by boiling and addition of DTT (1,4-Dithio-DL- threitol) . A strong bond was observed at Mw 90.000 and a shadow at Mw 100.000. When no boiling with DTT was performed the SDS-analysis resulted in a bond at Mw 170.000 indicating the molecular weight of the intact protein. The fact that the Mw of the intact protein is 170.000 is in accordance with the fact that the α-galactosidase was contained in the front fraction obtained from the gelfiltration analysis, in that the Mw of proteins contained in the front fraction would be expected to be higher than 150.000. It can thus be concluded that the α-galactosidase enzyme from A. niger described herein is a dimer of two protein chains each having a molecular weight of about 90.000. EXAMPLE 2 Preparation and characterization of α-galactosidase peptides Chemical degradation of a purified α-galactosidase prepara- tion with surplus CNBr was carried out in 70% HCOOH for 24 h at 25°C. Enzymatic degradation using chymotrypsin was carried out in 0.05 M NH 4 HC0 3 , 2 M urea for 5 h at 37°C at an enzyme: substrate ratio of 1:40 (w:w) . Peptides were purified using microbore reversed phase HPLC employing either C4 or C18 col¬ umns eluted with linear gradients of 75% aqueous 2-propanol in 0.1% aqueous TFA (triflouroacetic acid). Purified peptides were sequenced using an Applied Biosystems 473A protein sequencer. The following two peptides were obtained from chemical degra¬ dation with CNBr: CNBr-peptide l: Gly-Ala-His-Leu-Ser-Ala-Val-Pro-Asn-Ala-Gln-Thr-Gly-Arg-Thr- Val-Pro-Ile-Thr-Phe-Arg-Ala-His-Val- (SEQ ID No. 4) CNBr-peptide 2 : Asp-Asp-Gly-Trp-Phe-Gly-Asp-Lys-Tyr-Pro-Arg-Val-Ser-Asp-Asn- Ala-Gly-Leu-Gly-Asp-Asp- (SEQ ID No . 5) The following peptides were obtained from the enzymatic de¬ gradation using chymotrypsin: Chymotrypsin-peptide l: Thr-Thr-Arg-Phe-Pro-Asp-Val-Leu-Trp (SEQ ID No. 6) Chymotrypsin-peptide 2: Thr-Ser-Asp-Asn-Thr-Asp-Ala-Ile-Asp-Arg-Ile-Thr-Ile-Gln-Phe (SEQ ID No. 7) Chymotrypsin-peptide 3: Arg-Leu-Arg-Leu-Pro-Gln-Asp-Ser-Gln-Trp-Pro-Ala-Ala-Leu-Phe (SEQ ID No. 8) Chymotrypsin-peptide 4: Gly-Leu-Giu-Leu-Asp-Pro-Ala-Thr-Val-Glu-Gly-Asp-Glu-Ile-Val- Pro-Glu-Leu (SEQ ID No. 9) Chymotrypsin-peptide 5: Val-Met-Asp-Asp-Gly-Trp-Phe-Gly-Asp-Lys-Tyr-Pro-Arg-Val-Ser- Asp-Asn-Ala-Gly- (SEQ ID No. 10) It may be noted that amino acid residues 3-19 of the chymotrypsin-peptide 5 are present in CNBr-peptide 2 (amino acid residues 1-17) . EXAMPLE 3 Cloning of an Jεpergillus niger α-galactosidase Generation of an α-galactosidase probe As noted in Example 2 above chymotrypsin-peptide 5 and CNBr- peptide 2 are overlapping. Together they reveal the peptide: VMDDGWFGDKYPRVSDNAGLGDD (SEQ ID No. 11) Polymerase chain reaction (PCR) primers were designed in order to amplify the DNA sequence encoding this peptide sequence. In the 5' end (sense strand) the following degenerate primer was used: 5' TTACTAGTNATGGAYGAYGGNTGGTT 3' (5'#1: 64 species) (SEQ ID No. 12) . A Spe I site (ACTAGT) was anchored in the 5' end of this primer. In the 3' end (sense strand) the following degenerate primers were used: 5' TTGAGCTCRTCNCCYAANCCNGCRTT 3' (3'#1: 512 species) (SEQ ID NO. 13) . 5' TTGAGCTCRTCNCCNAGNCCNGCGTT 3' (3'#2: 512 species) (SEQ ID No. 14) 5' TTGAGCTCRTCNCCNAGNCCNGCATT 3' (3'#3: 512 species) (SEQ ID NO. 15) 5'#1 was used together with either 3'#1, 3'#2 or 3'#3 Genomic DNA was prepared from A. niger (ATCC 16882) as described by Leach et. al., 1986. This DNA was used as template in the PCR reactions (0.05 μg 5 genomic DNA, 100 pmol of each degenerate primer, 200 μM of dATP, dCTP, dGTP and dTTP, 1.5 mM MgCl 2 , 50 mM KC1, 0.01% ge¬ latine, 10 mM Tris-HCl pH 8.3 in a total volume of 100 μl) , and the following PCR program was run: 10 94°C for 2 min. , 1 cycle (0.5 μl of amplitaq' taq polymerase (Perkin Elmer - Cetus) was added during this incubation) . 94°C for 1 min., 50°C for 1 min., 72°C for 2 min., 30 cycles. 15 72°C for 5 min., 1 cycle. The products of the PCR amplifications were concentrated and run on an agarose gel. In the amplifications employing 3'#1 and 3'#3 no product except for 'primer dimer' was seen, but in the amplification employing 3'#2 a distinct fragment of 20 approx. 80 bp. was seen. This fragment was isolated, digested with the restriction enzymes Spel and Sad and ligated to the vector pUC19 (Yanish-Perron et al., 1985) digested with Xbal and Sad. The ligation mixture was transformed into Escheri- chia coli MC 1000 (Casadaban et al., 1980) made rm + by con- 25 ventional methods. Plasmid DNA isolated from a transformant was sequenced using the Sequenase kit (United States Biochemicals) following the manufacturers instructions. The sequence showed that the clo- 30 ned PCR fragment actually encoded the peptide fragment de¬ scribed above. The insert (86bp) of this plasmid was used as a probe in order to clone the α-galactosidase gene. Labelling of the probe 35 A radioactive labelled probe was prepared in the following way: 5 μg of the plasmid was digested with EcoRI and Sail and the 86 bp fragment was isolated from an agarose gel and dis¬ solved in 20 μl water. This was used as a template in a PCR reaction including 2 μl of the fragment, 50 pmol primer 5'#1, 50 pmol primer 3'#2, 10 pmol α 32 PdATP (3000 Ci/ mol) (DuPont NEG-012H) , 10 pmol dTTP, 10 pmol dCTP, 10 pmol dGTP, 1.5 mM MgCl 2 , 50 mM KC1, 0.01% gelatine, 10 mM Tris-HCl pH 8.3 in a total volume of 100 μl. The following temperature cycling program was run: 94°C for 2 min., 1 cycle (0.5 μl of amplitaq' taq polymerase (Perkin Elmer - Cetus) was added during this incubation) . 94°C for 1 min., 50°C for 1 min., 72°C for 2 min., 30 cycles. 72°C for 5 min., 1 cycle. The labelled fragment was isolated using a Sephadex G50 spun column as described by Maniatis et. al. (Maniatis et al., 1982) . The probe was heat denatured for 5 min, 100°C, and then added to the hybridization mixture. Genome cloning of the α-galactosidase Genomic DNA from A. niger was prepared as described above, and digested with various restriction enzymes, and the diges¬ tions were used for Southern blot analysis using the described α-galactosidase probe. A 4.5 kb BamHI fragment hybridized to the probe. This frag¬ ment was cloned the in the following way: A. niger genomic DNA was digested with BamHI, and fragments of 4-5 kb were isolated from an agarose gel. The fragments were ligated to pUC19 digested with BamHI and dephosphorylated with calf intestine alcaline phosphatase. The ligation mixture used to transform E. coli using ampicillin selection. 5000 clones were screened for the 4.5 kb α-galactosidase fragment by colony hybridization using the described α-galactosidase probe, and hybridizing clones were selected. Sequence analysis using the primers 3710 and 3711 of plasmid DNA isolated from one of these clones confirmed that the cloned fragment contained an α-galactosidase encoding sequence. This plasmid was termed pCaHj409. Sequence deduced from the M13 universal primer (United States Biochemicals) revealed that the 3' end of the gene was missing. 2178 bp of the insert covering the cloned part of the α-ga¬ lactosidase gene was sequenced from both strands using vari- ous primers. 3710 5' GCGTTATCGGACACTCG 3' (SEQ ID No. 16) 3711 5' GTTTGGGGACAAGTACC 3' (SEQ ID No. 17) cDNA cloning by PCR mRNA was prepared by guanidinium thiocyanate extraction fol¬ lowed by centrifugation in cesium chloride solution as described by Sambrook et. al, 1989, using fresh mycelium. First strand cDNA was synthesized from an oligo dT primer using the BRL superscript cDNA kit following the manufactu¬ rers instructions. The cDNA gene was cloned as a 5' fragment and a 3' fragment using the rapid amplification of cDNA ends (RACE) method as described by Frohman, 1990. The primer 3710 was used as a sequence specific primer for amplification of the 5' end, and 3711 was used as a sequence specific primer for amplification of the 3' end. In both cases the primers 2010 and 4433 were used as hybrid oligo dT primer and adaptor primer, respectively. 2010 5' CTGCAGTCGACTCTAGAGGATCCGCGGCCGCTTTTTTTTTTTT TTTTTTTTTTTT 3' (SEQ ID No. 18) 4433 5' TTACTGCAGTCGACTCTAGAGGATCCGCG 3' (SEQ ID No. 19) Composition of PCR reaction mixtures and the cycling profiles were as described by Frohman, op cit. The obtained 430 bp 5' fragment was digested with BamHI and Xhol and ligated to pUC19 digested with BamHI and Sail. The ligation mixture was transformed into E. coli using ampicil¬ lin selection. A plasmid containing an insert was sequenced from both strands using various primers. The sequence con¬ firmed that the fragment was an α-galactosidase cDNA frag- ment. The obtained 1300 bp 3' fragment was digested with Xhol and Xbal and ligated to pUC 19 digested with Sal I and Xba I. The ligation mixture was transformed into E. coli using ampicil- lin selection. A 1300 bp insert from a plasmid was confirmed to be an α-galactosidase fragment by sequence analysis from both strands using various primers. This plasmid was termed pCaHj 410. The genomic sequence and the cDNA sequence are shown in SEQ ID Nos. 1 and 2, respectively. The nucleotide fragments 302- 371, 628-716, 978-1032 of the genomic sequence represent in- tron sequences. The α-galactosidase protein sequence showed about 30% homology to the E. coli α-galactosidase encoded by the gene rafA (Aslandis et al., 1989). EXAMPLE 4 Expression of the α-galactosidase Construction of α-galactosidase expression vectors The plasmid pCaHj 409 was digested with Sal I and Pst I, and a 1.5 kb fragment was isolated and ligated to pUC 19 digested with Sal I and Pst I. After transformation into E . coli and isolation of plasmid, the resulting plasmid was digested with Sal I and EcoR I, and the 4.2 kb fragment was isolated. pCaHj 410 was digested with EcoR I and Sal I, and the 0.8 kb frag¬ ment was isolated and inserted into to the 4.2 kb fragment described above. The resulting plasmid was termed pCaHj 412. This plasmid was digested with ApaL I, the 3' recessed ends were filled in using the Klenow polymerase, and after phe¬ nol/chloroform extraction the mixture was digested with Hind III. The resulting 2.2 kb fragment was isolated. The Aspergillus expression plasmid pToC 68 (described in WO 91/17243) was digested with Bgl II, the 3' recessed ends were filled in using the Klenow polymerase, and after phenol/chlo¬ roform extraction the mixture was digested with Hind III. The 4.6 kb fragment was isolated and ligated to the 2.2 kb frag¬ ment described above. The resulting plasmid, termed pCaHj 413, contained a part of the aglN gene fused to the termina¬ tor of the amyloglycosidase gene of A. niger (Tamg) . The con¬ struction of pCaHj 413 is summarized in Fig. 1. pCaHj 413 was digested with Hind III and Xho I, and the 4.1 kb fragment was isolated. pCaHj 409 was digested with Hind III and Xho I, and the 4.0 kb fragment containing the 5' end of the aglN gene was isolated and ligated to the pCaHj 413 fragment. The resulting expression plasmid, termed pCaHj 414, contained the aglN promotor followed by the aglN gene fused to the AMG terminator. The construction of pCaHj 414 is sum¬ marized in Fig. 2. pMT 1560 (4169 bp) was derived from pHD 414 (described in WO 92/16634) by replacing the 617 bp BamH I - EcoR I fragment of pHD 414 with the BamH I - EcoR I digested PCR fragment ob¬ tained from a PCR reaction using pHD 414 as a template and the primers: 5'GCTCCTCATGGTGGATCCCCAGTTGTGTATATAGAGGATTGAGGAAGGAAGAGAAGTG- TGGATAGAGGTAAATTGAGTTGGAAACTCCAAGCATGGCATCCCTTGC 3' 106 mer (SEQ ID No. 20) , and 5'TGTTCTGGCTGTGGTGTACAGG 3 ' 22mer (SEQ ID No. 21) . pMT 1560 was digested with Nco I and Hind III, and the 3.9 kb fragment was isolated. pCaHj 414 was digested with Nco I and Hind III, and the 5.2 kb fragment containing the aglN gene was isolated and inserted into the 3.9 kb pMT 1560 fragment. The resulting plasmid was termed pCaHj 419. This plasmid was digested with Hind III and Xho I and the 5.2 kb containing the TAKA promotor of A. oryzae and the 3' end of the aglN gene fused to the AMG terminator was isolated. pCaHj 414 was used as a PCR template together with the primers 3710 and 4982 (containing a Hind III site followed by the ATG start codon of the aglN gene) : 3710 5' GCGTTATCGGACACTCG 3' (SEQ ID No. 16) 4982 5' GCAAGCTTTATCATCACCACCATGAT 3' (SEQ ID No. 22) The PCR conditions were as described in Example 3 above (in "Generation of an α-galactosidase probe"). The PCR fragment was digested with Hind III and Xho I and inserted into the 5.2 kb pCaHj 419 fragment. The resulting expression plasmid was termed pCaHj 424 and contained the aglN gene fused to the TAKA promotor in the 5' end and to the AMG terminator in the 3' end. The construction of pCaHj 424 is summarized in figure 3. Transformation of A . oryzae The plasmid pCaHj 414 was transformed into Aspergillus ory¬ zae IFO 4177 using selection on acetamide by cotransformation with pToC 90 as described in WO 91/17243. By cultivation in shake flasks or in submerged tank fermenta¬ tion of the cotransformants activity was accumulated in the broth. pCaHj 424 was transformed into A. oryzae IFO 4177 using the same method. Cotransformants expressed significantly higher amounts of α-galactosidase than pCaHj 414 transformants. Purification of α-galactosidase The culture supernatant from fermentation of Aspergillus or¬ yzae expressing the recombinant enzyme is centrifuged and filtered through a 0.2μm filter to remove the mycelia. 35-50 ml of the filtered supernatant (30-60 mg α-galactosi¬ dase) are ultrafiltrated in a Filtron ultracette or Amicon ultrafiltration device with a 10 kDa membrane to achieve 10 fold concentration. This concentrate is diluted 100 times in 25 mM Tris pH 8.0 in two successive rounds of ultrafiltration in the same device. This ultrafiltrated sample is loaded at 1.5 ml/min on a Pharmacia HR16/20 Fast Flow Q Sepharose anion exchanger equilibrated in 25 mM Tris pH 8.0. After the sample has been applied, the column is washed with two column vol¬ umes 25 mM Tris pH 8.0, and bound proteins are eluted with a linear increasing NaCl gradient from 0 to 0.6 M NaCl in 25 mM Tris pH 8.0. α-galactosidase elutes at approximately 0.25-0.3 M NaCl, but the enzyme in this fraction is not completely pure (approximately 80% purity) . Thus, the α-galactosidase containing fractions were concentrated by ultrafiltration in Amicon ultrafiltration device with a 10 kDa membrane to a volume of 4.5 ml and applied to a HR 26/60 Sephacryl S200 gelfiltration column in 0.25 M amonium acetate pH 5.5 at a constant flow of 1 ml/min. α-galactosidase is eluted as one distinct peak with a purity of approximately 90%. In order to achieve material purified to electrophoretic homogeneity, the α-galactosidase containing fractions are pooled, and ultra¬ filtrated into 10 mM sodium phosphate pH 6.8. The sample is applied onto a 8 ml BioRad HTP hydroxyl apatite column (10 mm internal diameter) at a constant flow rate of 1 ml/min. Bound enzymes are eluted by increasing the sodium phosphate concen¬ tration from 10 mM to 0.2 M over 40 min. α-galactosidase elutes at approximately 0.1 M sodium phosphate, and is more than 95% pure in this fraction. EXAMPLE 5 Characterization of α-galactosidase The following properties of the α-galactosidase expressed in and purified from A . oryzae were determined by the methods described in the Materials and Methods section above. The results obtained can be summarized in the following table: Mw 95 kDa pH-optimum 6.0 stability in water very stable temperature stability < 60°C in BSA for 1 hour temperature stability < 70°C in presence of raffinose specific activity towards (μmol/mg enzyme/min) a) PNP-α-galactosidase 90 b) raffinose 145 (100) c) stacchyose (350) d) guar gum (0) inhibition by galactose No transferase activity No Results in brackets are calculated from the HPLC results. pH optimum The pH optimum which is seen in Fig. 4 shows that the enzyme is most active at pH 6 but retains some activity in the whole range from pH 4-8. This is surprising in that the enzyme iso¬ lated from A. niger has a pH optimum in the range of 4-6. Degradation of stacchyose and raffinose and HPLC analysis From the HPLC chromatograms in Fig. 5 and 6 it is seen that degradation of raffinose (peak 4) is completed within 24 hours the reaction products being sucrose (peak 39, galactose (peak 1) and small amounts of fructose (peak 2) . The degrada¬ tion of stacchyose results in the formation of raffinose (peak 4) , sucrose (peak 39 and galactose (peak 1) . After 24 hours all stacchyose and raffinose has been converted into sucrose, galactose and small amounts of fructose. It was surprisingly found that the enzyme was not inhibited by galactose. REFERENCES CITED IN THE SPECIFICATION Leach et. al., 1986, Fungal gent, newsl., 33, 32-33. Yanish-Perron et al.. Gene 33, 103-119 (1985) Casadaban et al., J. Mol. Biol., 138, 179-207 (1980) Maniatis et al.. Molecular cloning, A laboratory manual. Cold Spring Harbor laboratory 1982 Sambrook, E. F. Fritsch and T. Maniatis. (1989) , Molecular cloning, a laboratory manual. 2. edition. Cold Spring Harbor Laboratory press) M. A. Frohman (1990) . RACE, rapid amplification of cDNA ends, in: M. A. Innis et. al. PCR protocols, A guide to methods and applications. Academic press) . Aslandis et al., 1989, Nucleotide sequences and operon struc¬ ture of plasmid borne genes mediating uptake and utilization of raffinose in Escherichia coli . J. Bact. 171, 6753-6763 WO 90/14101 (AEK DEVELOPMENT CORPORATION, US) US 3,846,239 of 5 Nov. 1974 (MONSANTO COMPANY) Worthington et al., "α-Galactosidase Activity of Fungi on Intestinal Gas-Forming Peanut Oligosaccharides", J.Agr.Food Chem., Vol. 22, No. 6 (1974) US 4,431,737 of 14 Feb. 1984 (ANIC, S.p.A., IT) Cruz and Park, (1982) , "Production of Funggal α-Galactosidase and Its Application to the Hydrolysis of Galactooligosacchar- ides in Soybean Milk", J. of Food Science - 1973, Vol. 47 Zapater et al. (1990) , Preparative Biochemistry, 20 (3&4) , pp. 263-296 Bahl and Agrawal (1969), "Glycosidases of Aspergillus niger", J. of Biol. Chem., Vol. 244 (11), pp. 2970-2978 Christakopoulos et al. (1990) , Process Biochemistry Interna¬ tional, pp. 210-212 Chun and Lee (1988) , Korean J. Food Sci. Technol. Vol. 20 (1), pp. 79-84 Jung and Lee (1986), Korean J. Food Schi Technol., Vol. 18 (6) , pp. 450-457 Lee and Wacek (1970) , "Galactosidases from Aspergillus niger", Archives of Biochem. and Biophysics 138. pp. 264-271 Adya and Elbein (1976), J. of Bact. , Vol. 129 (2), pp. 850- 856 Kaneko et al. (1991), Agric. Biol. Chem. 5_5 (1), pp. 109-115 Rackis, J. J. (1975), ACS Symposium Series 15, "Physiological Effects of Food Carbohydrates", pp. 207-222 Bahl et al., "α-Galactosidase, 3-Galactosidase, and j8-N-Ace- tylglucosaminidase from Aspergillus niger" (1972) , Methods in Enzymology, Vol 28, pp. 728-734 Overbeeke et al., 1990, Applied and Environmental Microbiol¬ ogy, Vol: 56, No. 5., pp. 1429-1434. Lipman and Pearson, 1985, Science 227, p. 1435 Hudson et al, 1989, Practical Immunology, Third edition, Blackwell Scientific Publications. Beaucage and Caruthers, 1984, Tetrahedron Letters 22, 1981, pp. 1859-1869 Matthes et al., 1984, The EMBO J. 3, 1984, pp. 801-805 R.K. Saiki et al., 1988, Science 239, pp. 487-491 Olsen and Adler-Nissen, Sonderdruck aus ZFL - Zeitschrift fur Lebensmittel-Technologie und Verfahrenstechnik 31. Jahrgang 1980 - Heft 8 - (Teil I); 32. Jahrgang 1981 - Heft 2 - (Teil II) Eriksen S., J. Food Sci. 48(2): 445-557, 1983. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: NOVO NORDISK A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: DENMARK (F) POSTAL CODE (ZIP) : DK-2880 (G) TELEPHONE: +45 44448888 (H) TELEFAX: +45 4449 3256 (I) TELEX: 37304 (ii) TITLE OF INVENTION : A. niger alpha-galactosidase (iϋ) NUMBER OF SEQUENCES: 22 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2476 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus niger (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTAGT∞CAG OC_AαπTGGG G_-ft__GAGZ_AG TQGGGGIGCC AAGGOGGAfJT GGGGGKXGKT 60 GOX3_3-3-ftG AAACTQGftlA ∞CT∞GAIG TllUIXX-GA 1GCM3ICG2G AOOSIODGGG 120 Q_IAAAAGGC OGGIGAGAGG AAGAACTCCT CK3TCACICA CCIGOCACAT GIGITAGGAT 180 TG_AO-X3_CAG C-A---_A_EC-_rC AOCAOCAIGA TO-X-TCITCC CATCCIGIGG ΕΞCCIGGGOC 240 TUTJ-AOSTT ATAαSGICAT TXπGCAGACA CX-CCα-CAAC T_GGσiTICA AACCCAO-GA 300 GIAIGGACIG σαGGGGGGGA GGAΪGCIACΓ GC_?VAC-GCTIG ATCΠCATIC GGAGtEAAGCT 360 GAOCAAACAG OGMαJETAC GAAlGGCACr _¥_T_?TααG__r TCAACGGOGA <_AA_KJK-ICA 420 TAiα_ATICC AIGTCAACAG TA-XAOCXΞGC GACTIGATIT CTX3__?0-ITT TOGIGGIGTC 480 GTCICOGGCA C_V-_XECTIC GCX3__TGGAA (X3GCIGTCA AOGGC-IGGGJT <_GGX_AIGOCT 540 GG1O-&A__CC GO_G3GAGIT OCXXXSAOCAA GGOCXΞ?IGGQG ATΓT X-GCAT αXXDGOO-JIT 600 O_TA__T0GGG AAaXEGCAGG TI_A-_ACTG_T MXX30CΣCC A3ATGIGICG <_ΑCGAGGIGA 660 TOGAAGGIAA AAATGCTTIG COOCXSGOCIG CXπGCX_ACAT TTOGOGAΪGC GCftGGCIGIC 720 ACAACTΠTGG TM3ICCKΣCT GI_A__GACAAC TA-__¥_CT00G TOGCGGOCGA CITGTCATAC 780 TCX_A-D_T_TC Cϊ_AAA_3-IGA TSOSA-tCGIG AGGAGIGTCA A1GIGATCAA a_AGGCO0CA 840 GG__V_EATCA CI3-ICGAGGC (XTIGCAAGC ATAA__l_3_rCG ATrTCXXCIA TGAAGAOCTC 900 GA(__¥D3CTCA GCCTOOGAGG OGACIQQGOC -_____-_3GCAA A-ECTICAGAG AAGO_AAGIG 960 <_AG___K_GOS TCX-AGGGGEA AGTCAGCATA GC____AAAAOC GACA__GGTGA OTIGCIGAC 1020 GGGAGAGEAG i-IT EGAAGC i_2[__CIOGAT -_ITCX_TCICA CCIICACAAT CCXTICCπG 1080 X_A1A-_T_AGA TCX_AGCTACT AO-EAATOGC AAGGOGAGGC ATOGGGTITC AACXTIGIAT 1140 ATAGCEGCTC TITCTOGGOC (_AAG_EGAGA AAGGAΪCGCA AGGΠTCAOC OQGGOGCIGC 1200 TOQGCTICAA C0O3GACCAA TJ_A LU_1GGA AO--?TOG00C TQGOGAGACT TIAAL IΑR 1260 O_GAGIGIGΓ TGCAGTCIAC TOGS-CA G GOCΓΓGGCIC _V__?GICICGC AAATΓOCAOC 1320 GGC_-A__K_CG C__AOCA -CTC AIGAAGAGCΆ AG3?iπ_CCAC GαXXEAOOSG CX-3GITCIGC 1380 TCAATAGCIG GGAAGGIGIT TA__TICGACT ACAATCAAAG CMX30CCΑA ACKTTCOαG 1440 w-AG xxsc TGCXCTGGCT GICCADCICT TIGICAIGGA CGΆCCGCIGG TITCGGGACA 1500 AG__A X3CIOG A3IGT0C-GAT AAOGOCX3GAC IGGX_CGACIG GMGOOCAAT C ΑG GCCCI 1560 TCCX-GGA03G GTTIGACCC-X GTOGIGCAAG ACATCACAAA TC-TCACXCTC AATOGCACAG 1620 ACTCCACAAA ACTK-GCITT GGTATITQGG TGGAGOOOΞA GAIGGTCAAC (_0-_AA__TCCA 1680 CTCTCTAOCA CX__AACAO0αG GAGIGGGCGC TI MGCCGG GCXTTACCOC <_GIACCGAGC 1740 GTOaGAAOCA GCTICGIOCTC AAOCIGGCGC T_XXX3GC_GT GCAGGACITC ATCAΪAGACT 1800 TCATGAOGAA CXTGTEACAA GA-EAD03GCA TI-XETAOST CAAAIGQGAC AACAACCGGG 1860 GAATACAOGA GAO___X-CTCT CX-t_TOSACIG AOS-I BGEA C__IGCriGGC CTCTAGOGGG 1920 TCaTCX_ACAC ACIGACCAOC αSC lOOJ-G -_IGTOCTGIG GGAAGGAIGT GCXSCQGGIG 1980 GAGGOOSCIT IGAIGCTGGC AIGCTGCAGT ATCK3CX-CCA GATCTCGACT TCXEATAACA 2040 CX3GACX-CCAT OGAOOGAATC ACXftTOCAAT KIGGAOCTC GCTTGCCIAC CC3GO_ATCAG 2100 <_2_AIGGGIGC <_X_ACXTCICC G0 -3_?ICC_-A A03CACAGAC OQCTOGCACr GKXXX3-ITA 2160 C3?TIOCGOΞC ACACCTIGCΓ ATCaiGGGIG GTK3TTCX3G CT]?TOGAGCIG GATC03GOGA 2220 αSGIGGAAGG GGAOGAAAIA GHODCGAGC TTCTIGOSCT GGOQGAAAAA GIG OOCTA 2280 TCA1TITGAA OIGAGATCIG TA__O3GCIAC C3<XTAOCTCA ___A -TOOCAG TO -CCTGCAG 2340 CACT TTIUΓ GACTCAGGAT GXX)3CACAGG ciυ iciυ r C_-_ACΠCAGG T X_AGCOGAA 2400 TCTCAAOCAT G03G03IGGG TC___GCIGCT GGGGTIGGAC CTAAGGOGAC TAa-AOC-TIGA 2460 TGGAGATAAG CATCCG 2476 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2028 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: A. niger ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATCAΪCGGTC TICα-S-IGCr GIGGIGCX-TG GGO ITITJ-A 0-. __---_AOQG TC_ATTCIGCA 60 GAC-AOGOOOG <__AACIGGGGT TK3-AAO0CA C-AGAOGATCG T___-GAAIGG C_ACTAGITTC 120 α-A-πGA OG GO__AGAAIGΓ CTCAIATOGA TKX_A_KΠCA ACAGEAOCAC CX_GCX_ACITG 180 A_LT1CTIL_ATC A-_T_?IGΪIGG IGTϋGI TOC GGCAl_AA-_OC Cπ EOCAGr GGAACCTGCT 240 GTCAAOGGCT GGGJICGGCAT GCCIGGTCGA ATOCECX_GGG A ,T111XXJ-_A α_AAGGO0Sr 300 GGGGA ΠCC Gca-[α_ααGc u_. ιu_-ι__rr c _AAiOGG CRGGI AIAC TGJCTGICACA 360 ACTITQGTAG _XX3__C3GI_- TGACAACEAT AGCIC05IOG <_GGCCGACIT GTCAΪACICC 420 ATA TIUJΑ AATAIGAIGC G_-KX_TGAGG #__X_TCAA_IG TGATCAAOCA GGGGOCAGCT 480 AAIϊ-KS-CTA TOGAGGOOCT TGCAAGCA-CA AGE-TOGATT TOCOCIATGA AGAOCTOGAC 540 _ΗGG-X_AGOC TCπSAGGOGA CIGGGOCAGA GAGGCAAATC T -AGAGAAG C5W X3C G 600 TAIGGOSTOC AGGGA__TO_G AAGCAGEACT GGATAΠCCT <_r_X_AOCITCA CAATCXX IC 660 CITGOCAXAG TAGA OC-AGC TACTAGOGAA TOGCAAGGOG AGGCAIGGGG TTICAAXTT 720 GTEATATAOOG GCTCΠTTCTC GGOOCAAGIA GAGAAAGGAT αGC_-AGGITT (-ACXXDGGGOG 780 CT_CT03GCT TftAOOCGGA ( XMσm∑ G TGGAAOCTIG GαXTGGOGIA GACTITAACT 840 τσooσα-AGr UIUTIUCAGΓ CTAC OGGAC AAAGGCCΠG GCT aGIGI TOGCAAAITC 900 C-AD03GCIAT ___OGCAAOCA <XTC-_TGAAG ___GAAGTIOG CCΑ03ICCGA OCCGUL-JJIT 960 CrcCIGAAIA GCT3GGAAGG lUlTJMTIC GACTACAATC AAAGCAGCAT OSAAACTCTT 1020 GCOGAAGAGT α_GCIGCXXT GGCTGICCAC CTTI'IUICA TGGACGACEG Cπ_CTITGGG 1080 GACAAGIACC CIOGAGK3IC CX-AIAAO-CC GGACIQGGOG ACIQGAIGOC <__A-_K_CAGGG 1140 CGCTTGOOGG AOGGGTIGAC σCXXΞGPOGIG C_AAGAGATCA C-AAA-TCTCAC 03IC__A__GGC 1200 AC_V3_3ieCA CSAAACFICG CTΠGGTAΪT TGGGIGGAGC OOGAGAIQGT CS XX-CAAT 1260 TCX_ACICTCT ACCACGAACA α-OGGAGIGG GOGCITCAIG CXXSGGOCTEA O-XX30G__AGC 1320 GAGOGTOGGA ACX_AGCTCGT (XTTCAAOCIG Gα_X_TKX_GG CTGTCCAGGA CTTCAICATA 1380 GACTTCATGA OGAAOCT3IT ACAAGAIACC GGC_A__TICCT AOITCAAATG GGACAACAAC 1440 033GGAAIAC AOGAGAOGCC CTCTOOGTOG ACTCAOCATC AG__-_-__GCT TGGCCTCTAC 1500 CXΞGGΠGΠΌG ACACACIQAC CAO_X3GCΠC CCGGAIGICC TGTGGGAAGG AIGΓGCCICG i560 GGIGGAGGOC GCITTGAIGC TGGCAIGCIG CAG__A_IGTOC (_t_X_AGATCIG GACTICCGAT 1620 AACAODGAOG α_A_ OGAQOG AA__CAO-_ATC C-V-_?TTGGGA CCTOGCTIGC CTAGOCGOCA 1680 TC_AGCAA_[QG GTCOαCAOCT CTOOGOGGIT OCΓAACGCAC AGADOGGIΌG CACTGIGCOC 1740 A-CTACTπCC GOGCACAαGT TCCTAIGAΓG GGIQGITCIT TOSGCTIGGA GCIGGAICOG 1800 GCX_-_CX_GIGG AAGGGGACGA AATAGΠCOC GAGCITCTIG OGCIGG03GA AAAAGIGAAC 1860 CXΠATCATIT TGAAOGGAGA TCTGΓATOGG CTACGOCIAC CTCAAGACTC CX_AGIGGGCΓ 1920 G<_AGC-A(_TCT TTGTCACTCA GGATGGOGCA CSV-GC3GTIC TC3TCTACIT (3_3GICCAGC 1980 αSAATCTCAA CXZAIGOQGOG TGGGTCAGGC TGCIQGGGIT GGADCTAA 2028 ( 2 ) INFORMATION FOR SEQ ID NO : 3 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 676 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear ( ii) MOLECULE TYPE : protein ( iii ) HYPOTHETICAL : NO ( iii) ANTI-SENSE : NO (v) FRAGMENT TYPE : internal (Vi) ORIGINAL SOURCE : (A) ORGANISM: A. niger (Xi) SEQUENCE DESCRIPTION : SEQ ID NO : 3 : Met lie Gly Leu Pro Mat Leu Trp cys Leu Gly Leu Fhe Thr Leu iyr 1 5 10 15 Gly His Ser Ala Asp Thr Pro Ala Ihr Gly Val Ser Asn Pro Gin Thr 20 25 30 lie Val Thr Asn Gly Thr Ser Phe Arg Leu Asn Gly Asp Asn Val Ser 35 40 45 Tyr Arg Fhe His Val Asn Ser Thr Thr Gly Asp Leu lie Ser Asp His 50 55 60 Fhe Gly Gly Val Val Ser Gly Thr He Pro Ser Pro Val Glu Pro Ala 65 70 75 80 Val Asn Gly Trp Val Gly Met Pro Gly Arg He Arg Arg Glu Phe Pro 85 90 95 Asp Gin Gly Arg Gly Asp Phe Arg He Pro Ala Val Arg He Arg Glu 100 105 110 Ser Ala Gly Tyr Thr Ala Val Thr Thr leu Val Val His Leu Tyr Asp 115 120 125 Asn Tyr Ser Ser Val Ala Ala Asp Leu Ser Tyr Ser He Ehe Pro Lys 130 135 140 Tyr Asp Ala He Val Arg Ser Val Asn Val He Asn Gin Gly Pro Gly 145 150 155 160 Asn He Thr He Glu Ala Leu Ala Ser He Ser He Asp Phe Pro Tyr 165 170 175 Glu Asp Leu Asp Met Val Ser Leu Arg Gly Asp Trp Ala Arg Glu Ala 180 185 190 Asn Val Gin Arg Ser Lys Val Gin Tyr Gly Val Gin Gly Fhe Gly Ser 195 200 205 Ser Thr Gly Tyr Ser Ser His Leu His Asn Pro Phe Leu Ala He Val 210 215 220 Asp Pro Ala Thr Thr Glu Ser Gin Gly Glu Ala Trp Gly Phe Asn Leu 225 230 235 240 Veil Tyr Thr Gly Ser Ehe Ser Ala Gin Val Glu Lys Gly Ser Gin Gly 245 250 255 Fhe Thr Arg Ala Leu Leu Gly Fhe Asn Pro Asp Gin Leu Ser Trp Asn 260 265 270 Leu Gly Pro Gly Glu Thr Leu Thr Ser Pro Glu Cys Val Ala Val Tyr 275 280 285 Ser Asp Lys Gly Leu Gly Ser Val Ser Arg Lys Fhe His Arg Leu Tyr 290 295 300 Arg Asn His Leu Met Lys Ser Lys Fhe Ala Thr Ser Asp Arg Pro Val 305 310 315 320 Leu Leu Asn Ser Trp Glu Gly Val Tyr Fhe Asp Tyr Asn Gin Ser Ser 325 330 335 He Glu Thr Leu Ala Glu Glu Ser Ala Ala Lsu Gly Val His Leu Fhe 340 345 350 Val Met Asp Asp Gly Tφ Phe Gly Asp Lys Tyr Pro Arg Val Ser Asp 355 360 365 Asn Ala Gly Leu Gly Asp Trp Met Pro Asn Pro Ala Arg Lai Pro Asp 370 375 380 Gly Leu Thr Pro Val Val Gin Asp He Thr Asn Leu Thr Val Asn Gly 385 390 395 400 Thr Glu Ser Thr Lys Leu Arg Phe Gly He Trp Val Glu Pro Glu Met 405 410 415 Val Asn Pro Asn Ser Thr Leu Tyr His Glu His Pro Glu Tip Ala Leu 420 425 430 His Ala Gly Pro Tyr Pro Arg Thr Glu Arg Arg Asn Gin Leu Val Leu 435 440 445 Asn Leu Ala Leu Pro Ala Val Gin Asp Fhe He He Asp Phe Met Thr 450 455 460 Asn Leu Leu Gin Asp Thr Gly He Ser Tyr Val Lys Trp Asp Asn Asn 465 470 475 480 Arg Gly He His Glu Thr Pro Ser Pro Ser Thr Asp His Gin Tyr Met 485 490 495 Leu Gly Leu Tyr Arg Val Fhe Asp Thr Leu Thr Thr Arg Fhe Pro Asp 500 505 510 Val Leu Trp Glu Gly Cys Ala Ser Gly Gly Gly Arg Fhe Asp Ala Gly 515 520 525 Met Leu Gin Tyr Val Pro Gin He Trp Thr Ser Asp Asn Thr Asp Ala 530 535 540 He Asp Arg He Thr He Gin Fhe Gly Thr Ser Leu Ala Tyr Pro Pro 545 550 555 560 Ser Ala Met Gly Ala His Leu Ser Ala Val Pro Asn Ala Gin Thr Gly 565 570 575 Arg Thr Val Pro lie Thr Fhe Arg Ala His Val Ala Met Met Gly Gly 580 585 590 Ser Phe Gly Leu Glu Leu Asp Pro Ala Thr Val Glu Gly Asp Glu He 595 600 605 Val Pro Glu Leu Leu Ala Leu Ala Glu Lys Val Asn Pro He He Leu 610 615 620 Asn Gly Asp Leu Tyr Arg Lai Arg Lai Pro Gin Asp Ser Gin Trp Pro 625 630 635 640 Ala Ala Leu Phe Val Thr Gin Asp Gly Ala Gin Ala Val Leu Phe Tyr 645 650 655 Phe Arg Ser Ser Arg Met Ser Thr Met Arg Arg Gly Ser Gly Cys Trp 660 665 670 Gly Trp Thr Glx 675 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Gly Ala His Leu Ser Ala Val Pro Asn Ala Gin Thr Gly Arg Thr Val 1 5 10 15 Pro He Thr Phe Arg Ala His Val 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Asp Asp Gly Trp Fhe Gly Asp Lys Tyr Pro Arg Val Ser Asp Asn Ala 1 5 10 15 Gly Leμ Gly Asp Asp 20 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Thr Thr Arg Phe Pro Asp Val Leu Trp 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Thr Ser Asp Asn Thr Asp Ala He Asp Arg He Thr He Gin Hie 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Arg Leu Arg Leu Pro Gin Asp Ser Gin Tip Pro Ala Ala Leu Phe 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Gly Leu Glu Leu Asp Pro Ala Thr Val Glu Gly Asp Glu He Veil Pro 1 5 10 15 Glu Leu (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear ( ii) MOLECULE TYPE : peptide ( iii) HYPOTHETICAL: NO ( iii) ANTI-SENSE : NO (v) FRAGMENT TYPE : internal (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 10 : Val Met Aεp Aεp Gly Ttp Fhe Gly Aεp Lys Tyr Pro Arg Val Ser Aεp 1 5 10 15 Asn Ala Gly (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Val Met Asp Asp Gly Trp Fhe Gly Asp Lys Tyr Pro Arg Vil Ser Asp 1 5 10 15 Asn Ala Gly Leu Gly Aεp Asp 20 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic DNA primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TTACIAGINA TGGAYGAYGG NIGGTT 26 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic DNA primer (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TIGAGCTCRΓ CNC-CYAANCC GCKΓT 26 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (Vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic DNA primer ( XI ) SEQUENCE DESCRIPTION : SEQ ID NO : 14 : TTGAGCTCRΓ CNCCNAI-NOC NGOGΓΓ 26 ( 2 ) INFORMATION FOR SEQ ID NO : 15 : ( I ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ϋi) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 15 : T GAGCTCKT CNCCNZ_-NCC NGCAIT 26 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GCΪ_1TATCX_G ACACTOS 17 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GTTTGGGGAC AAGTAOC 17 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CIGCBGICGA OXΠAGAGGA oαGOGGcαG CTTTTTTT Γ TTTTTTΠTT TTTTT 55 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (Vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TTACTGGAGT CX-ACTCIAGA GGATOαGOG 29 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 20 : GCTOCTCAIG GTOGATOCCC AG_?TCIGTAT ATAGAGGA T GAGGAAGGAA GAGAAGIGIG 60 GAIAGAGGTA AATTGAGITG GAAACTOCAA GCS-IGGCATC OCTTGC 106 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TGΓKIΓGGCΓ GIGGTGTACA GG 22 (2) INFORMATION FOR SEQ ID NO: 22: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GCAAGCITTA TC-AICAOCAC CAIGAT 26

Claims: CLAIMS 1. A DNA construct comprising a DNA sequence encoding a poly- peptide having α-galactosidase activity, wherein the DNA se¬ quence a) encodes a polypeptide comprising the amino acid sequence shown in the appended SEQ ID No. 3, or b) is an ana¬ logue of the DNA sequence of a) , which i) hybridizes with the DNA sequence shown in the appended SEQ ID No. 1 or 2 or an oligonucleotide probe prepared on the basis of said DNA sequence or on the basis of the amino acid sequence shown in SEQ ID No. 3 under the conditions defined herein, ii) encodes a polypeptide reactive with an antibody reacting with at least one epitope of a polypeptide comprising the a- mino acid sequence shown in the appended SEQ ID No. 3, and/or iii) encodes a polypeptide being at least 50% identical with the polypeptide having the amino acid sequence shown in the appended SEQ ID No. 3. 2. A DNA construct according to claim 1, wherein the DNA se- quence is derived from a microorganism, a plant or a mammal. 3. A DNA construct according to claim 1 or 2, wherein the DNA sequence is derived from a bacterium or a fungus. 4. A DNA construct according to any of claims 1-3, wherein the DNA sequence is derived from a strain of Aspergillus. especially from a strain of A. niger. 5. A DNA construct according to any of the preceding claims, in which the DNA sequence encodes an α-galactosidase having a pi in the range of 4.0-5.5 as determined by IEF as described herein, a pH optimum in the range of 5.0-7.0 determined under the conditions described herein, a temperature optimum within of about 170.000 Da, and/or a specific activity of above about 250 GALU/mg protein. 6. A DNA construct according to any of claims 1-5, wherein 5 the DNA sequence is as shown in the appended SEQ ID No. 1 or 2. 7. A DNA construct according to any of the preceding claims in which the DNA sequence is a cDNA sequence, a genomic DNA 10 sequence or a synthetic DNA sequence or a mixed cDNA, genomic and/or synthetic DNA sequence. 8. A recombinant expression vector comprising a DNA construct according to any of claims 1-7. 15 9. A cell comprising a DNA construct according to any of claims 1-7 or a vector according to claim 8. 10. A cell according to claim 9, which is a microbial cell. 20 11. A cell according to claim 10 which is a bacterial cell, a yeast cell, or a fungal cell. 12. A cell according to claim 11, in which the bacterial cell 25 is a cell of a gram-positive bacterium such as Bacillus or Streptomyces or a cell of a gram-negative bacterium such as Escherichia. the yeast cell is a cell of Saccharomyces. and the fungal cell is a cell of Aspergillus. 30 13. An α-galactosidase preparation encoded by a DNA construct according to any of claims 1-7. 14. A process for producing an α-galactosidase enzyme or a variant thereof exhibiting α-galactosidase activity, compris- 35 ing culturing a cell according to any of claims 9-12 in a suitable culture medium under conditions permitting express¬ ion of the α-galactosidase enzyme or the variant, and reco¬ vering the resulting enzyme or variant from the culture. 15. Use of an α-galactosidase preparation as claimed in claim 13 for the hydrolysis of an α-galactoside to galactoses and sucroses. 5 16. The use according to claim 15, in which the α-galactoside is present in composition prepared from legumes, nuts, seeds, grains, cereals or vegetables. 17. The use according to claim 16 for the .in vivo conversion 10 of α-galactoside-linked sugars in mammals. 18. The use according to claim 17 for pre-treatment of food or feed containing α-galactosides. 15 19. Use of the α-galactosidase preparation according to claim 13 as an digestive aid. 20. A food or feed comprising an α-galactosidase preparation according to claim 13. 20 21. A method of preparing an enzyme-modified soy bean product comprising subjecting a composition containing the soy bean product to be modified to enzymatic treatment in the presence of an α-galactosidase preparation according to claim 13. 25 22. A method of converting a soy bean product comprises a) inserting a DNA construct according to any of claims 1-7, optionally present in a suitable expression vector, into a suitable host organism, 30 b) culturing the host organism in a suitable culture me¬ dium under conditions permitting expression of the poly¬ peptide encoded by the DNA construct, and recovering the resulting polypeptide from the culture, and 35 c) subjecting a composition containing the soy bean pro¬ duct to be modified to enzymatic treatment in the pres¬ ence of the polypeptide recovered in step b) .

Inventors: HALKIER TORBEN, HJORT CARSTEN M, KNAP INGE HELMER, KOFOD LENE VENKE, HALKIER, TORBEN, HJORT, CARSTEN, M., KNAP, INGE, HELMER, KOFOD, LENE, VENKE
Applicants: HALKIER TORBEN, HJORT CARSTEN M, KNAP INGE HELMER, KOFOD LENE VENKE, NOVONORDISK AS, HALKIER, TORBEN, HJORT, CARSTEN, M., KNAP, INGE, HELMER, KOFOD, LENE, VENKE, NOVO NORDISK A/S
Application year: 1994
Main classification: C12N9
IPC codes: A23L1, A61K38, A61P1, C07H21, C12N1, C12N9, C12N15, C12P19, C12R1